siRNA inhibition of aspartyl-asparaginyl β-hydroxylase expression impairs cell motility, Notch signaling, and fetal growth

Aspartyl-asparaginyl-β-hydroxylase (AAH) regulates cell motility and invasiveness by enhancing Notch signaling. Invasive trophoblastic cells, which mediate placentation, normally express high levels of AAH. Previously, we showed that ethanol-impaired placentation is associated with reduced AAH expression. The present study determines the degree to which inhibition of AAH expression is sufficient to impair functions required for placentation.Immortalized, first trimester-derived, human trophoblastic cells (HTR-8/SVneo) were transfected with siRNA targeting AAH (siRNA-AAH) or no specific sequences (siRNA-Scr) using the Amaxa electroporation system. Directional motility was measured using an ATP luminescence-based assay. For in vivo studies, we microinjected siRNA-AAH or siRNA-Scr directly into the implantation sites (mesometrial triangle) of gestation-day-17, Long Evans pregnant rats, and harvested placentas 24 h later for histologic and molecular studies.siRNA-AAH transfection reduced AAH expression and directional motility in HTR-8/SVneo cells. In vivo delivery of siRNA-AAH reduced AAH expression and mean number of invasive trophoblastic cells at the implantation site. These adverse effects of siRNA-AAH were associated with impaired fetal growth and significantly reduced expression of Notch-signaling network genes.AAH is an important, positive regulator of trophoblastic cell motility, and inhibition of AAH in vivo leads to impaired implantation and fetal growth, and alters Notch-signaling mechanisms, similar to the effects of chronic ethanol exposure.